Activation of the complement system (C system) represents an important mechanism in the immune defence. At the same time it represents a double-edged weapon because on the one hand guarantees protection of the host but on the other is capable of damaging tissues where complement is activated by several pathological circumstances. The increased susceptibility to bacterial infections and autoimmune disorders observed in patients with inherited deficiencies of the C system clearly demonstrates the particular importance of this system in host protection against infectious agents and in the clearance of immunocomplexes.
These protective functions result from complement activation in cascade fashion that generates biologically active products. Some of those, such as C1q, C3b and C3bi, opsonize the infectious agents enabling their disposal. Instead others, such as C5a and C5b67, have the function to recruit phagocytic cells at the site of inflammation or lyse sensitive targets as in the case of the membrane attack complex (MAC). Unfortunately, these molecules, once they are produced, are not able to discriminate between endogenous and exogenous targets, and would provoke serious damage to tissues and cells if those were not protected by potent membrane or extracellular inhibitors acting at various levels in the complement activation cascade. However, the action of inhibitors is overwhelmed in presence of a massive activation of the C system, in serious infectious diseases or autoimmune disorders, and the activated complement causes tissue and cell destruction.
The C5a fragment and the C terminal complex (TCC) are among the products involved in tissue destruction in several inflammatory processes. Above normal levels of these activation products can be found in synovial fluid of rheumatoid arthritis patients and in cerebrospinal fluid of patients with several diseases of the central nervous system. Elevated C5a levels have been found also in polytraumatised patients as well as in patients with damage from myocardial ischaemia and re-perfusion.
Therefore the role of C5a in development of these diseases is now recognised.
This is demonstrated by the signs of pulmonary stress, hypotension and leukopenia shown by animals that have received intravenous injection of this anaphylatoxin. Moreover bronchial instillation of C5a is capable of inducing strong inflammatory reactions in rabbit lung.
TCC is generated from the C5b fragment released by enzymatic cleavage of C5 by the action of C5 convertase. Recently it has been demonstrated that TCC induces inflammation because of tissue damage deriving from its lytic activity and of numerous non-cytotoxic effects on phagocytes and other cell types.
TCC has been identified in several tissues in diverse pathological conditions, including rheumatoid arthritis, glomerulopathies multiple sclerosis, demyelinating peripheral neuropathies, atherosclerosis and myocardial infarction. The development of experimental animal models of these diseases with selective deficiencies in late C components has further contributed to define the role of these components in development of tissue damage.
Because of the fundamental role played by C5a and TCC in promoting chronic inflammation and tissue damage, several attempts have been made to neutralise the late components of the C complex as therapeutic strategy to prevent these complications in diseases associated with C5 activation. This molecule turns out to be an ideal therapeutic target, since its neutralisation inhibits the late sequence of activation events of the cascade, without interfering with the opsonizing activity of the early components of this cascade. Mouse monoclonal antibodies specific for human, mouse and rat C5 and capable of inhibiting the production of C5a and of the membrane attack complex (MAC) are already commercially available. Anti-C5 antibodies have been successfully used in mice to prevent the development of collagen-induced arthritis and to improve the clinical curse of glomerulonephritis, and in rats to reduce myocardial ischemia and re-perfusion.
In the last years two single chain antibodies (single chain antibody or single chain fragment variable, scFv) have been produced that are both described in patent application WO 95/29697. These antibodies are able to penetrate tissues more rapidly than the whole antibody. The first single chain antibody obtained by assembling variable regions of a mouse antibody for C5 retains the ability of the original antibody to inhibit assembly of the MAC and partially block C5a production (Evans, M. J et al, 1995, Mol. Immunol. 32:1183). Moreover, this antibody is able to prevent C5b-9 deposits in the heart re-perfused with human plasma or in heart insufficiency. The second scFv is a humanised mouse antibody anti-C5, that is obtained by inserting murine CDR regions (complementarity determining region) in the structure of the variable region of human light and heavy chains. This antibody (Thomas, T. C. et al., 1996, Mol. Immunol. 33:1389) is able to inhibit C5a and C5b-9 formation although the recognised epitope mapped around amino acids 860-865 of the C5 molecule and corresponding to peptide KSSKC [SEQ ID NO: 36], turns out to be far from the C5 convertase cleavage site. Moreover subsequent studies (Fitch, J. C. et al., 1999, Circulation 100:2499) have demonstrated that this antibody is able to inhibit complement hemolytic activity, to attenuate myocardial damage, cognitive damage and postoperative haemorrhage in a group of patients with cardiopulmonary bypass.